Conditionally Immortalized Mouse Gonadotropes

描述（由申请方提供）：促性腺激素合成和 调节，特别是涉及卵泡刺激素（FSH）的调节， 将大大受益于在培养中有现成的促性腺激素供应， 准确反映体内促性腺激素功能。在过去，分散 初级促性腺激素难以纯化， 此外，转化的促性腺细胞系可能不能完全反映 由于它们来自转化的细胞， 处于未知的发展阶段在此，申请人提出， 从永生鼠体内分离促性腺激素ImmortoMouse的所有细胞 可以表达已知的不耐热的SV 4 O大T抗原（TLLTag）， 转化小鼠促性腺激素。ImmortoMouse中分离的促性腺激素 在允许的条件下（33 ℃，干扰素-γ）应迅速分裂 但在39 ℃时恢复为完全分化的促性腺激素， TLLTag。许多高度分化的细胞类型已经被扩增， 这种策略，并增殖了50多代，而没有损失 分化ImmortoMouse促性腺激素的有效分离将涉及 用H2Kk细胞表面抗原标记它们，然后使用 磁性抗H2KK微珠。该手术的成功最近被 在申请人的实验室中使用来自 使用绵羊FSH β启动子独特靶向H2Kk的转基因小鼠 到促性腺激素如果条件永生化导致某些促性腺激素 去分化，这些细胞可以通过随后的分离消除， 因为H2Kk模拟FSH产生。所有促性腺细胞系 将监测FSH、LH和促性腺激素受体的产生 释放激素（GnRH）以及卵巢类固醇激素，GnRH， TGF-β和其他TGF-β家族成员，如激活素。这些细胞系 将提供给生殖生物学界进行研究 促性腺激素的调节和合成。

DESCRIPTION (provided by applicant): Studies of gonadotropin synthesis and regulation, especially those involving the follicle-stimulating hormone (FSH), would benefit greatly by having a ready supply of gonadotropes in culture that accurately reflect gonadotrope function in vivo. In the past, dispersed primary gonadotropes have been prohibitively difficult to purify for such studies; also, transformed gonadotrope cell lines may not fully reflect characteristics of adult gonadotropes since they come from cells transformed at unknown stages of their development. Herein the applicant proposes isolating gonadotropes from the ImmortoMouse. All cells of the ImmortoMouse can express a thermolabile SV4O large T antigen (TLLTag) already known to transform mouse gonadotropes. Isolated gonadotropes from the ImmortoMouse should divide rapidly under permissive conditions (33 C with interferon-gamma) but revert to fully differentiated gonadotropes at 39 C which destabilizes the TLLTag. Numerous highly differentiated cell types have been amplified using this strategy and proliferated more than 50 generations without loss of differentiation. Effective isolation of ImmortoMouse gonadotropes will involve tagging them with the H2Kk cell surface antigen followed by isolation using magnetic anti-H2Kk-microbeads. The success of this procedure has recently been established in the applicant's laboratory using dispersed pituitary cells from transgenic mice that use the ovine FSH beta promoter to target H2Kk uniquely to gonadotropes. If conditional immortalization causes some gonadotropes to dedifferentiate, these cells can be eliminated by subsequent isolation based on H2Kk selection since H2Kk mimics FSH production. All gonadotrope cell lines will be monitored for production of FSH, LH and receptors for gonadotropin releasing hormone (GnRH) as well as regulation by ovarian steroids, GnRH, inhibin and other TGF beta family members such as activin. These cell lines will be available to the reproductive biology community for studies on gonadotropin regulation and synthesis.